Method for manufacturing protease

ABSTRACT

A method for manufacturing protease which comprises culturing Bacillus sp. 0-20 belonging to the genus Bacillus in a culture medium to produce protease and collecting the protease from the medium.

ilite States Patent Tani et al.

[ 1 July4, 1972 [54] METHOD FOR MANUFACTURING PROTEASE [72] Inventors:Isamu Tanl, Tokunhima-shi; Masatoml Otuului, Naruto-shi;, YoshlyesuOsakl, 'lokushimwken; Takuo Haglno, Narutoshi, all of Japan [73]Assignee: ()tsuka Kagaku Yakuhin Kabushiki Kaisha, Osaka-shi, Japan [22]Filed: Oct. 22, 1969 21 Appl. No.: 868,613

[56] References Cited FOREIGN PATENTS OR APPLICATIONS 701,1 l9 0/1953England ..l95/66 OTHER PUBLICATIONS Primary ExaminerA. Louis MonacellAssistant ExaminerRoger B. Andewelt Att0rneyLarson, Taylor and Hinds [57] ABSTRACT A method for manufacturing protease which comprisesculturing Bacillus sp. 0-20 belonging to the genus Bacillus in a culturemedium to produce protease and collecting the protease from the medium.

4 Claims, No Drawings METHOD FOR MANUFACTURING PROTEASE This inventionrelates to a method for manufacturing protease, and more particularly toa method for producing protease by the culture of a specific novelmicro-organism belonging to the genus Bacillus.

A main object of the invention is to provide protease having high orderof enzymatic activity and a method for preparing the same. The above andother objects of the invention which will be apparent from thedescription to follow, can be attained by culturing Bacillus sp. -20belonging to the genus Bacillus in a culture medium to produce proteaseand collecting the protease thus produced from said medium.

The present invention is based on a new discovery that said Bacillussp.,0-20 is capable of producing protease having an extremely high orderof enzymatic activity as compared with other publicly known strainsbelonging to the genus Bacillus.

The Bacillus sp. 0-20 used in the invention is a novel variant preparedby irradiating with ultraviolet light a certain kind of a rod which isakin to the Bacillus cereus disclosed in Japanese Journal ofBacteriology" Vol. 23, No. 2 (1968), pages 145- l 5 l. The presentstrain, Bacillus sp. 0-20, has been deposited with deposition numberPERM-P No. 270 in Fermentation Research Institute of Agency ofIndustrial Science and Technology, Japan, since Feb. 20, I969.

The mycological properties of this strain are as follows:

21. Condition of growth in medium 1. Size: 1.0-1.2 p. X 5.0-5.2 p. 2.Form: oval with angular and 3. Spore: formed, oval, 1.0 p. X 2.0-2.2 p.4. Mobility: Yes, having peritrichous 5 Degree of growth on each mediuma. Liquid culture Bouillon: slightly turbid with sediment, forming thinfilm ring. Turbidity is not cleared.

Peptone water: evenly turbid forming a faint ring.

b. Stab culture Gelatine medium: rapidly liquifies.

c. Plate culture Gelatin medium: hydrolyzed in a wide range.

d. Slant culture Bouillon-agar medium: Grow briskly, lustrous andgreyish white in color, spread, wetted and slightly swollen.

Glucose-bouillon-agar medium: Grow briskly, greyish white in color, wetluster, spread, wetted and slightly denser than the bouillon-agarmedium.

Potato slice: Grow briskly, wet and creamy white in color.

6. Condition of surface of colony: generally spherical and homogeneous,rough berries, grey white in color.

7. Color of strain: grey white in color.

8. Pathogenic property: not recognized.

. Physiological property 1. Conditions for growth: pH 6.8-7.2 is theoptimum, temperature 30-32 C is the optimum and no growth at 50 C and isaerobic.

2. Gram s stain: Positive 3. Acid fastness: 4. Methyl red test:

5 Voges Proskauer test:

6. Production of indole:

7. Production of ammonium: 8. Reduction of nitric acid salt: 9.Production of catalase: 10. Liquefaction of gelatin and casein: l 1.Production of hydrogen sulfide: l2. Hydrolysis of starch: r.

13. Utility of citric acid:

4. Mannose: 5. Fructose: 6. Galactose: 7. Maltose:

8. Succarose: 9. Trehalose: l0. Raftinose: ll. Sorbit:

l2. Glycerol: l3. lnosite: 14.Salycine: 15. a-methylglucoside: l6.Lactose: l 7. lnulin:

l8. Dextrine: l 9. Starch: 20. Cellulose:

The taxological positionof the present strain was determined bycomparing the mycological properties thereof with those of otherpublicly known bacteria in accordance with the method described inBergeys Manual of Deterrninative Bacteriology (7th edition) with thefollowing result.

Class: Schizomycetes Order: Eubacteriales Family: Bacillaceae Genus:Bacillus As mentioned above the strain of the invention rapidlyliquefies in the gelatine stab culture, and by lightly dying theprotoplasm it looks vacuolar, but in voges Proskauer reaction it showspositive and mobile. In these respects it differs from Bacillus cereusvar; mycadies. Further, the present strain produces no acid fromarabinose and xylose and it is akin to the Bacillus cereus in thegrowing condition and physiological property. However, the presentstrain is obviously novel strain which is akin to but different fromBacillus cereus in view of the facts that it shows somewhat bettergrowing condition in the glucose-nitric acid salt medium and that it hasno properties to reduce nitric acid salt and to produce acid fromstarch, while Bacillus cereus generally produces acids from glycerol andsuccarose. Moreover, the strain used in the invention is found to bedifferent from the strain KP 931 which is akin to Bacillus cereus andwhich was disclosed in Joumal of the Agricultural Chemical Society ofJapan Vol. 40, No. 6 (1966), pages 252-256, in the production of acidsfrom arabinose, glycerose and sucearose, and also to be different fromthe strain akin to Bacillus cereus which was reported in JapaneseJournal of Bacteriology Vol. 23, No. 2 (1968), pages -151, in theproduction of hydrogen sulfide andof acids from lactose, mannose andsuccarose. From the above findings the strain used in the invention wasascertained to be novel strain belonging to the genous Bacillus andbeing a variant akin to Bacillus cereus, and was newly named Bacillussp. 0-20.

The medium used in the invention may be prepared in the conventionalmanner with glucose, fructose, galactose, maltose and other sugars as acarbon source, and with soybean cake, coan steep liquor and like as anitrogen source. There may be added an inorganic salt, such as potassiumdihydrogenphosphate, potassium chloride, etc. and/or a vitamin, asrequired. The pH of the medium may be in the range of 5.0 to 9.0,preferably 6.8 to 7.0.

According to the method of the invention the Bacillus sp. 0-20 isinoculated in the medium and the strain is cultured at a temperature of30 to 38 C for 12 to 72 hours in accordance with shaking oraeration-agitation culture method known in the art, thus producingprotease. The resultant protease thus produced and accomodated in themedium may be separated and purified by various known methods, such asprecipitation, salting out or dialysis method; When the culture mediumused is dilute, concentration with ion-exchange resin or under reducedpressure may also be applied.

For better understanding of the invention exam les are given below, inwhich enzymatic activity [PU] f' g -of the l. resultant proteaseproducing a non-protein substance showing folin color corresponding to l'y of tyrosine in one minute is shown as one unit of the enzymaticactivity, when the protease to be tested is treated in milk casein baseat 30 C for minutes and absorbance of trichloroacetic acid-solublesubstance is measured at a wave length of 660 mp.

EXAMPLE 1 ln 1,000 ml of water were dissolved 1.4 g of Nag-IP0 1.0 g ofKH PO 2.0 g of NaCl, 0.1 g of M,SO 6.0 g of sodium glutamate and 10.0 gof glucose to produce a medium of pH 7.0, and the medium was thensterilized. Bacillus sp. 0-20 was inoculated in the resultant medium andcultured with shaking at 30 C for 3 days. After the strain used wascentrifugally separated from the cultured product, the enzymaticactivity of the supernatant fluid was measured by casein-folincalorimetry B method mentioned before. The enzymatic activity, [PU] 3;jg"- per ml was 1050.

For comparison the strain which is akin to Bacillus cereus and wasdisclosed in Japanese Journal of Bacteriology Vol. 23, No. 2 (I968),pages 145-151, was cultured in the same manner as in Example 1. Theenzymatic activity per ml of the supernatant fluid obtained was measuredwith the result of W1 i fi y-M215.

EXAMPLE 2 EXAMPLE 3 In this example was used a medium of pH 7.0containing 150 g of glucose, 5,000 g of alkali extract containing 500 gof defatted bean cake extract, 50 g of KH PO and g of CaCO: dissolved in10 liters of water. After the medium was sterilized Bacillus sp. 0-20was inoculated in the medium and cultured by aeration-agitation methodat 37 C for 17 hours at a rate of aeration of 1 liter per liter of themedium and at a rotation rate of 300 r.p.m. From the resultant culturedproduct the strain was centerifugally separated and the proteaseactivity per ml of the slipernatant fluid thereof was measured, whichshowed PU Q- of 6,000 sulfate at 5C to salt out the protease. The saltedout product was dissolved in 0.02 mole of calcium Said supernatant fluidwas saturated with ammonium sulfate at 5 C to salt out the protease. Thesalted out product was dissolved in 0.02 mole of calcium acetate, andthen dialyzed overnight at 3 C with 0.02 mole of calcium acetate. To thedialyzed liquid was further added acetone 0.6 time the volume of theliquid, and after viscous precipitate produced was removed, acetone wasfurther added thereto in an amount 2.5 times the volume of the liquid.The precipitate thus produced was frozen to dryness, whereby a crudeenzyme product was obtained. From 1 liter of the supernatant fluid wasobtained 800 g of protease in a powder form. The enzymatic activity permg of the resultant protease was measured with the result of [PU] 5;fiof 1050.

What we claim is:

l. A method for manufacturing protease which comprises culturingBacillus sp. FERM-P No. 270 belonging to the genus Bacillus in a culturemedium to produce protease and collecting the protease from the medium.

2. The method for manufacturing protease according to claim 1, in whichpH of said culture medicum is in the range of 5.0 to 9.0.

3. The method for manufacturing protease according to claim 2, in whichpH of said culture medium is in the range of 6.8 to 7.0.

4. The method for manufacturing protease according to claim 1, in whichthe temperature of said culture medium is in the range of 30 to 38 C.

2. The method for manufacturing protease according to claim 1, in whichpH of said culture medicum is in the range of 5.0 to 9.0.
 3. The methodfor manufacturing protease according to claim 2, in which pH of saidculture medium is in the range of 6.8 to 7.0.
 4. The method formanufacturing protease according to claim 1, in which the temperature ofsaid culture medium is in the range of 30* to 38* C.